Thromboxane biosynthesis and future events in diabetes: the ASCEND trial

Abstract Background and Aims Thromboxane (TX) A2, released by activated platelets, plays an important role in atherothrombosis. Urinary 11-dehydro-TXB2 (U-TXM), a stable metabolite reflecting the whole-body TXA2 biosynthesis, is reduced by ∼70% by daily low-dose aspirin. The U-TXM represents a non-invasive biomarker of in vivo platelet activation and is enhanced in patients with diabetes. This study assessed whether U-TXM is associated with the risk of future serious vascular events or revascularizations (SVE-R), major bleeding, or cancer in patients with diabetes. Methods The U-TXM was measured pre-randomization to aspirin or placebo in 5948 people with type 1 or 2 diabetes and no cardiovascular disease, in the ASCEND trial. Associations between log U-TXM and SVE-R (n = 618), major bleed (n = 206), and cancer (n = 700) during 6.6 years of follow-up were investigated by Cox regression; comparisons of these associations with the effects of randomization to aspirin were made. Results Higher U-TXM was associated with older age, female sex, current smoking, type 2 diabetes, higher body size, urinary albumin/creatinine ratio of ≥3 mg/mmol, and higher estimated glomerular filtration rate. After adjustment for these, U-TXM was marginally statistically significantly associated with SVE-R and major bleed but not cancer [hazard ratios per 1 SD higher log U-TXM (95% confidence interval): 1.09 (1.00–1.18), 1.16 (1.01–1.34), and 1.06 (0.98–1.14)]. The hazard ratio was similar to that implied by the clinical effects of randomization to aspirin for SVE-R but not for major bleed. Conclusions The U-TXM was log-linearly independently associated with SVE-R in diabetes. This is consistent with the involvement of platelet TXA2 in diabetic atherothrombosis.


Urine extraction
Urine samples of 1 mL were thawed at 37°C and centrifuged at 340g for 10 minutes; 60 ul of acetic acid was added to reach a pH of approximately 3.5, 1,000 cpm 3 H-thromboxane B2 were added to 1 mL urine sample.Samples were loaded into a 1 mL/50 mg C18 column, prewashed with 1 mL absolute methanol and 2.5 mL distilled H2O.Column was then washed with 1.6 mL distilled H2O and 2 mL acetonitrile/water (15:85, vol/vol), dried and eluted with 2.5 mL isooctane/ethyl acetate (1:1, vol/vol).The eluate was loaded on a 1 mL/100 mg SiOH column prewashed with 2 mL isooctane/ethyl acetate (1:1,vol/vol), eluted with 2 mL ethylacetate/methanol (60:40, vol/vol).After drying, eluate was resuspended in 1 mL PBS/0.1% BSA buffer, 500ul of the final resuspension were counted for calculating the recovery.The ELISA measurements were corrected for the % of recovery based on 3 Hthromboxane B2 cpm counts.This method has been published before (2).For urine samples of <1ml volume, 0.5 mL were extracted, by adapting the above protocol as follows: 30 ul acetic acid were added to 0.5 mL urine, after drying, the eluate was resuspended in 0.5 mL PBS/0.1% BSA buffer, 250ul were counted for assessing recovery.
To assess the stability and reproducibility of the extraction procedure over the entire sub-study duration, a pool of urine from healthy donors was aliquoted into 1mL samples and frozen until use.In each experimental set, aliquots were thawed and spiked with vehicle (ethanol 0.05% vol/vol) or with known concentrations of the exogenous cold TXM commercial standard at final concentrations ranging from 0.5 and 2 ng/ml.These experiments were performed at least every two months over the entire duration of the analyses.

TXM measurements
Urinary TXM was measured in the extracted samples by a standard Enzyme Linked Immunosorbent Assay (ELISA) assay (2,3).Ninety-six-well plates were coated with commercial monoclonal anti-rabbit IgG antibodies according to the standard method for coating.TXM in urinary extracts was measured with a standard AchE ELISA immunometric method, using a specific rabbit polyclonal antibody (1).The ELISA assay using this antibody had a range of detection from 0.5 to 0.0039 ng/ml, a sensitivity calculated as B/B0 (Bound/Maximum Bound) 80% of 0.01 ng/ml, and an overall inter-assay coefficient of variation of 8.8%.Samples that measured <0.018 ng/ml were assayed with a different commercial anti-rabbit IgG, which had a lower range of detection from 0.25 to 0.0019 ng/ml and a 80% B/B0 of 0.004 ng/ml.The cross-reactivity of the anti-TXM antibodies against other prostanoids that can be measured in urines, namely PGE2 and the isoprostane 8-iso-PGF2alpha was <0.05%.
The validation of the method was based on the U.S. Food and Drug Administration guidelines for Validation of Bioanalytical Methods (4).Internal standards were used for intra-assay validation and consisted of pools of urinary extracts from healthy donors which were aliquoted, frozen and one aliquot was used per each plate.TXM final values were corrected for the concentration of urinary creatinine, that was measured by a commercial kit based on the Jaffe's reaction (5).Two internal standards were used in each assay plate: a pool of urine and a commercial standard.These internal standards were always included to assess the consistency and reproducibility of the assays over time.

Reproducibility of Urinary Extraction method
To assess the stability and reproducibility of the extraction procedure, we performed experiments with cold TXM spike during the entire study duration.Representative experiments are shown on Figure S1.Cold spike recoveries appeared quite consistent over time and independent of the endogenous levels of TXM in the spiked urine samples.

Reproducibility of ELISA measurements in urinary extracts
As part of the validation process, we repeated the ELISA assay in 320 urine extracts selected based on residual sample volume availability.The correlation between the two measurements is shown in Figure S2.

Reproducibility of the urine extraction procedure
We also assessed the reproducibility of the urine extraction procedure and ELISA measurements in 319 urinary samples selected based on the remaining available volume.The second extraction was always performed with 5 ml urine.A high correlation about the two measurements is represented in Figure S3.

Reproducibility of urinary creatinine measurements
Urinary creatinine was repeated in 163 samples, randomly selected based on the available remaining volume.The correlation between the two measurements was very high and shown in Figure S4.Urinary creatinine 2nd measurement (mg/ml)

Figure
Figure S1 Recovery of spiked 11-dehydro thromboxane B2 (TXM) standard from urine samples Figure S2 Reproducibility of ELISA measurements Figure S3 Reproducibility of the extraction method Figure S4 Reproducibility of urinary creatinine measurements Figure S5 Associations of aspirin versus placebo treatment allocation with outcomes in U-TXM quartiles.

Figure S1 .
Figure S1.Recovery of spiked 11-dehydro thromboxane B2 (TXM) standard from urine samples.Urine samples from healthy volunteers were spiked with exogenous TXM or vehicle, extracted by chromatography and analysed.The y-intercept corresponds to the amount of TXM in the vehicle-spiked urine sample.Each line represents the fitting of a representative experimental set at different study months.The two panels show different study months.

Figure S2 .
Figure S2.Reproducibility of ELISA measurements.In 320 random samples, urinary extracts were measured by the ELISA on two distinct occasions.The plot represents the values of 11-dehydro thromboxane B2 (TXM) of the 1 st and the 2 nd determination, the linear regression line and 95% confidence interval.TXM is expressed in pg/ml of urine.

Figure S3 .
Figure S3.Reproducibility of the extraction method.Extractions were repeated from 319 urine samples, and 11-dehydro thromboxane B2 (TXM) was measured in the repeated extracts.The plot represents the values of urinary TXM corrected by % recovery in the 1 st and in the 2 nd extraction, the linear regression line and 95% confidence interval.TXM is expressed in pg/ml of urine.

Figure S4 .
Figure S4.Reproducibility of urinary creatinine measurements.In 163 random samples, urinary creatinine analyses were assessed on two separate occasions.The plot represents the 1 st and the 2 nd determinations, the linear regression line and 95% confidence interval.

Figure S5 .
Figure S5.Associations of aspirin versus placebo treatment allocation with outcomes inU-TXM quartiles.Adjusted for basic factors and predictors of log U-TXM.P-value for trend over quartiles is calculated using the mean log U-TXM within each quartile.CI=confidence interval, N=number of participants.

Table S1 : Baseline characteristics by availability of a valid urinary 11-dehydro thromboxane B2 (U-TXM) measure in the present and previous ASCEND sub-studies Characteristic U-TXM study
*Parish S et al. 'Effect of low-dose aspirin on urinary 11-dehydro-thromboxane B2 in the ASCEND (A Study of Cardiovascular Events iN Diabetes) randomized controlled trial'.Trials (2023)